Canine herpesvirus (CHV) causes a fatal, hemorrhagic disease in neonatal puppies and a self-limiting, usually subclinical, upper respiratory tract infection in adult dogs (Appel, 1987). Little is known about the genomic structure of CHV. The genome has not been mapped and no nucleotide sequence has been published. In particular, genes encoding immunologically pertinent proteins have not been identified.
Herpesvirus glycoproteins mediate essential viral functions such as cellular attachment and penetration, cell to cell spread of the virus and, importantly, determine the pathogenicity profile of infection. Herpesvirus glycoproteins are critical components in the interaction with the host immune system (Spear, 1985a; Spear 1985b). Herpesvirus glycoproteins are antigens recognized by both the humoral and cellular immune systems and, have been shown to evoke protective immune responses in vaccinated hosts (Wachsman et al., 1987; Marchioli et al., 1987; Eberle et al., 1980; Papp-Vid et al., 1979).
During a herpesvirus infection, the majority of the immune response is directed against viral envelope glycoproteins. These antigens have been shown to elicit both humoral and cellular immune responses. Several reports have indicated that in other herpesvirus systems immunization with the herpesvirus gB, gC and/or gD glycoproteins can induce a protective immune response.
The well characterized glycoproteins of herpes simplex virus include gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL and gM (Spear, 1985a; Spear 1985b; Ackermann et al., 1986; Frink et al. 1983; Frame et al., 1986; Longnecker et al., 1987; Richman et al., 1986; Swain et al., 1985; Zezulak, 1984; Roizman and Sears, 1990; Hutchinson et al., 1992a; Hutchinson et al., 1992b; Baines and Roizman, 1993). A number of studies have indicated the importance of herpes simplex virus glycoproteins in eliciting immune responses. Hence, it has been reported that gB and gD can elicit important immune responses (Berman et al., 1983; Cantin et al., 1987; Cremer et al., 1985; Lasky et al., 1984; Martin et al., 1987a; Martin et al., 1987b; Paoletti et al., 1984; Perkus et al., 1985; Rooney et al., 1988; Wachsman et al., 1987; Zarling et al., 1986a; Zarling et al., 1986b). gC can stimulate class I restricted cytotoxic lymphocytes (Glorioso et al., 1985; Rosenthal et al., 1987) whereas gD can stimulate class II cytotoxic T cell responses (Martin et al., 1987a; Martin et al,. 1987b; Wachsman et al., 1987; Zarling et al., 1986a; Zarling 1986b). gG was shown to be a target for complement-dependent antibody directed virus neutralization (Sullivan et al., 1987; Sullivan et al., 1988). A number of glycoproteins from other herpesviruses have also been shown to elicit important immune responses.
Both subtypes of equine herpesvirus (EHV) express six abundant glycoproteins (Allen et al., 1986; Allen et al., 1987). The genomic portions of the DNA sequences encoding gp2, gp10, gp13, gp14, gp17/18, and gp21/22a have been determined using lambda gtll expression vectors and monoclonal antibodies (Allen et al., 1987). Glycoproteins gp13 and gp14 were located in the same locations within the L component of the genome to which the gC and gB homologs, respectively, of herpes simplex virus map (Allen et al., 1987). The envelope glycoproteins are the principal immunogens of herpesviruses involved in eliciting both humoral and cellular host immune responses (Ben-Porat et al., 1986; Cantin et al., 1987; Glorioso et al., 1984; Wachsman et al., 1988; Wachsman et al., 1989) and so are of the highest interest for those attempting to design vaccines.
Recently, the nucleotide sequence of the Kentucky T431 strain of the EHV-1 transcriptional unit encoding gp13 has been reported (Allen et al., 1988). The glycoprotein was shown to be homologous to the herpes simplex virus (HSV) gC-1 and gC-2, to the pseudorabies virus (PRV) gIII and the varicella-zoster virus (VZV) gpV (Allen et al., 1988). EHV-1 gp13 is thus the structural homolog of the herpesvirus gC-like glycoproteins.
The nucleotide sequence of EHV-1 gp14 (Whalley et al., 1989; Riggio et al., 1989) has recently been reported. Analysis of the predicted amino acid sequence of gp14 glycoprotein revealed significant homology to the corresponding glycoprotein of HSV, gB.
Monoclonal antibodies directed against some EHV-1 glycoproteins have been shown to be neutralizing (Sinclair et al., 1989). Passive immunization experiments demonstrated that monoclonal antibodies directed against gp13 or gp14 (Shimizu et al., 1989) or against gp13, gp14 or gp17/18 (Stokes et al., 1989) could protect hamsters against a lethal challenge. Other gB and gC glycoprotein analogs are also involved in protection against diseases caused by alphaherpesviruses (Cantin et al., 1987; Cranage et al., 1986; Glorioso et al., 1984).
Pseudorabies virus (PRV), an alphaherpesvirus, is the causative agent of Aujesky's disease. The PRV genome consists of a 90.times.10.sup.6 dalton double stranded DNA (Rubenstein et al., 1975) separated by inverted repeat sequences into unique long (U.sub.L) or unique short (U.sub.S) segments (Stevely, 1977; Ben-Porat et al., 1979). The PRV genome encodes approximately 100 polypeptides whose expression is regulated in a cascade-like fashion similar to other herpesviruses (Ben-Porat et al., 1985; Hampl et al., 1984).
PRV glycoprotein gp50 is the Herpes simplex virus type 1 (HSV-1) gD analog (Wathen et al., 1984). The DNA open reading frame encodes 402 amino acids (Petrovskis et al., 1986). The mature glycosylated form (50-60 kDa) contains O-linked carbohydrate without N-linked glycosylation (Petrovskis et al., 1986). Swine serum is highly reactive with PRV gp50, suggesting its importance as an immunogen. Monoclonal antibodies to gp50 neutralize PRV in vitro with or without complement (Wathen et al., 1984; Wathen 1985; Eloit et al., 1988) and passively protect mice (Marchioli et al., 1988; Wathen et al., 1985; Eloit et al., 1988) and swine (Marchioli et al., 1988). Vaccinia virus recombinants expressing PRV gp50 induced serum neutralizing antibodies and protected both mice and swine against lethal PRV challenge (Kost et al., 1989; Marchioli et al., 1987; Ishii et al., 1988).
PRV gIII is the HSV-1 gC analog (Robbins et al., 1986). Functional replacement of PRV gIII by HSVgC was not observed (Whealy et al., 1989). Although PRV gIII is nonessential for replication in vitro (Wathen et al., 1986; Robbins et al., 1986), the mature glycosylated form (98 kDa) is an abundant constituent of the PRV envelope. Anti-gpIII monoclonal antibodies neutralize the virus in vitro with or without complement (Hampl et al., 1984; Eloit et al., 1988; Wathen et al., 1986) and can passively protect mice and swine (Marchioli et al., 1988). The PRV glycoprotein gIII can protect mice and swine from lethal PRV challenge after immunization with a Cro/gIII fusion protein expressed in E. coli (Robbins, A., R. Watson, L. Enquist, European Patent application 0162738A1) or when expressed in a vaccinia recombinant (Panicali, D., L. Gritz, G. Mazzara, European Patent application 0261940A2).
PRV gpII is the HSV-1 gB homolog (Robbins et al., 1987). Monoclonal antibodies directed against PRV gpII have been shown to neutralize the virus in vitro (Ben-Porat et al., 1986) with or without complement (Wittmann et al., 1989). Moreover, passive immunization studies demonstrated that neutralizing monoclonal antibodies partially protected swine (Marchioli et al., 1988). Immunization with NYVAC (highly attenuated vaccinia virus)-based recombinants expressing pseudorabies virus (PRV) gII (gB) or gp50 (gD) has been shown to protect swine against a virulent PRV challenge (Brockmeier et al., 1993). Furthermore, vaccinia recombinants expressing PRV gII and gp50, or gII, gIII (gC) and gp50 have been shown to elicit a higher level of protection than recombinants expressing gII or gp50 alone, suggesting a potential synergistic effect with these glycoproteins (Riviere et al., 1992).
The herpes simplex virus type 1 (HSV1) genome encodes at least eleven antigenically distinct glycoproteins: gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL and gM (Roizman et al., 1990). Mice immunized with purified HSV1 gB, gC or gD are protected against lethal HSV1 challenge (Chan, 1983). Mice have also been protected against lethal HSV1 or HSV2 challenge by passive immunization with antibodies to total HSV1 (Davis et al., 1979) or HSV2 (Oakes et al., 1978) virus and with antibodies to the individual HSV2 gB, gC, gD or gE glycoproteins (Balachandran et al., 1982).
Vaccinia virus vectors expressing HSV1 gB (McLaughlin-Taylor et al., 1988) and HSV1 gC (Rosenthal et al., 1987) have been shown to induce cytotoxic T-cell responses. In addition, it has been shown that mice immunized with recombinant vaccinia virus expressing either HSV1 gB (Cantin et al., 1987), HSV1 gC (Weir et al., 1989) or HSV1 gD (Paoletti et al., 1984) are protected against a lethal challenge of HSV1. A recombinant vaccinia virus expressing HSV1 gD has also been shown to be protective against HSV2 in a guinea pig model system (Wachsman et al., 1987).
Bovine herpesvirus 1 (BHV1) specifies more than 30 structural polypeptides, 11 of which are glycosylated (Misra et al., 1981). Three of these glycoproteins, gI, gIII and gIV, have been characterized and found to be homologous to the herpes simplex virus (HSV) glycoproteins gB, gC and gD (Lawrence et al., 1986; Zamb, 1987). Immunization with purified bovine herpesvirus type 1 (BHV1) gI (gB), gIII (gC) and/or gIV (gD) has been shown to protect cattle against a BHV1/Pasteurella haemolytica challenge (Babiuk et al., 1987).
Feline herpesvirus type-1 (FHV-1) has been shown to contain at least 23 different proteins (Meas et al., 1984; Fargeaud et al., 1984). Of these, at least five are glycosylated (Fargeaud et al., 1984; Compton, 1989) with reported molecular masses ranging from 120 kDa to 60 kDa. The FHV-1 glycoproteins have been shown to be immunogenic (Meas et al., 1984; Compton, 1989). Like several other alphaherpesviruses, FHV-1 appears to have a homolog of glycoprotein B (gB) of HSV-1 (Maeda et al., 1992). The FHV-1 gB glycoprotein is a 134 kDa complex which is dissociated with B-mercaptoethanol into two glycoproteins of 66 kDa and 60 kDa. The FHV-1 DNA genome is approximately 134 Kb in size (Rota et al., 1986).
Epstein Barr Virus (EBV), a human B lymphotropic herpesvirus, is a member of the genus lymphocryptovirus which belongs to the subfamily gammaherpesvirus (Roizman et al., 1990). Since the EBV genome was completely sequenced (Baer et al., 1984) as the genomes of VZV (Davison et al., 1986), HSV1 (McGeoch et al., 1988), MCHV (Chee et al., 1990) and EHV1 (Telford et al., 1992) numerous homologies between these different herpesviruses have been described (Kieff et al., 1990).
Human cytomegalovirus (HCMV) is a member of the betaherpesvirinae subfamily (family Herpesviridae). Three immunologically distinct families of glycoproteins associated with the HCMV envelope have been described (Gretch et al., 1988): gCI (gp55 and gp93-130); gCII (gp47-52); and gCIII (gp85-p145). The gene coding for gCI is homologous to HSVI gB.
In addition, immunization with a fowlpox recombinant expressing Marek's disease virus (MDV) gB has been shown to protect chickens against a virulent MDV challenge (Nazarian et al., 1992).
The results of these studies indicate that an immune response against gB, gC and/or gD glycoproteins can protect target species animals against a herpesvirus challenge and, that the provision of nucleotides for CHV gB, gC and gD glycoproteins is a valuable advance over the current state of the art as it allows for the provision of the glycoproteins and, antigenic, immunological or vaccine compositions from the vector systems or from the glycoproteins. Further, the glycoproteins from expression of the nucleotides can be used to elicit antibodies which can be further used in antibody binding diagnostic assays, kits or tests for ascertaining the presence or absence in a sample such as sera of the glycoprotein(s) and therefore the presence or absence of CHV or of an immune or antigenic response (to either CHV or to the glycoproteins). Thus, many utilities flow from the provision of the nucleotides for CHV gB, gC and gD glycoproteins.
Various vector systems exist for the expression of exogenous DNA, such as the phage, e.g., lambda, and E. coli systems (Allen et al., 1987; Robbins, EPA 0162738A1; Panicali, EPA 0261940A2, each of which is expressly incorporated herein by reference).
Vaccinia virus and more recently other poxviruses have been used for the insertion and expression of foreign genes. The basic technique of inserting foreign genes into live infectious poxvirus involves recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus (Piccini et al., 1987).
Specifically, the recombinant poxviruses are constructed in two steps known in the art and analogous to the methods for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus described in U.S. Pat. Nos. 4,769,330, 4,772,848, 4,603,112, 5,100,587, and 5,179,993, the disclosures of which are incorporated herein by reference.
First, the DNA gene sequence to be inserted into the virus, particularly an open reading frame from a non-pox source, is placed into an E. coli plasmid construct into which DNA homologous to a section of DNA of the poxvirus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA containing a nonessential locus. The resulting plasmid construct is then amplified by growth within E. coli bacteria (Clewell, 1972) and isolated (Clewell et al., 1969; Maniatis et al., 1982).
Second, the isolated plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome respectively gives a poxvirus modified by the presence, in a nonessential region of its genome, of foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, particularly DNA from a non-pox source, that codes for gene products not ordinarily produced by the genome into which the exogenous DNA is placed.
Genetic recombination is in general the exchange of homologous sections of DNA between two strands of DNA. In certain viruses RNA may replace DNA. Homologous sections of nucleic acid are sections of nucleic acid (DNA or RNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene in the recombinant viral genome. Additional strategies have recently been reported for generating recombinant vaccinia virus.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions. First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed.
Vaccinia virus has been used successfully to immunize against smallpox, culminating in the worldwide eradication of smallpox in 1980. In the course of its history, many strains of vaccinia have arisen. These different strains demonstrate varying immunogenicity and are implicated to varying degrees with potential complications, the most serious of which are post-vaccinial encephalitis and generalized vaccinia (Behbehani, 1983).
With the eradication of smallpox, a new role for vaccinia became important, that of a genetically engineered vector for the expression of foreign genes. Genes encoding a vast number of heterologous antigens have been expressed in vaccinia, often resulting in protective immunity against challenge by the corresponding pathogen (reviewed in Tartaglia et al., 1990a).
The genetic background of the vaccinia vector has been shown to affect the protective efficacy of the expressed foreign immunogen. For example, expression of Epstein Barr Virus (EBV) gp340 in the Wyeth vaccine strain of vaccinia virus did not protect cottontop tamarins against EBV virus induced lymphoma, while expression of the same gene in the WR laboratory strain of vaccinia virus was protective (Morgan et al., 1988).
A fine balance between the efficacy and the safety of a vaccinia virus-based recombinant vaccine candidate is extremely important. The recombinant virus must present the immunogen(s) in a manner that elicits a protective immune response in the vaccinated animal but lacks any significant pathogenic properties. Therefore attenuation of the vector strain would be a highly desirable advance over the current state of technology.
A number of vaccinia genes have been identified which are non-essential for growth of the virus in tissue culture and whose deletion or inactivation reduces virulence in a variety of animal systems.
The gene encoding the vaccinia virus thymidine kinase (TK) has been mapped (Hruby et al., 1982) and sequenced (Hruby et al., 1983; Weir et al., 1983). Inactivation or complete deletion of the thymidine kinase gene does not prevent growth of vaccinia virus in a wide variety of cells in tissue culture. TK.sup.- vaccinia virus is also capable of replication in vivo at the site of inoculation in a variety of hosts by a variety of routes.
It has been shown for herpes simplex virus type 2 that intravaginal inoculation of guinea pigs with TK.sup.- virus resulted in significantly lower virus titers in the spinal cord than did inoculation with TK.sup.+ virus (Stanberry et al., 1985). It has been demonstrated that herpesvirus encoded TK activity in vitro was not important for virus growth in actively metabolizing cells, but was required for virus growth in quiescent cells (Jamieson et al., 1974).
Attenuation of TK.sup.- vaccinia has been shown in mice inoculated by the intracerebral and intraperitoneal routes (Buller et al., 1985). Attenuation was observed both for the WR neurovirulent laboratory strain and for the Wyeth vaccine strain. In mice inoculated by the intradermal route, TK.sup.- recombinant vaccinia generated equivalent anti-vaccinia neutralizing antibodies as compared with the parental TK.sup.+ vaccinia virus, indicating that in this test system the loss of TK function does not significantly decrease immunogenicity of the vaccinia virus vector. Following intranasal inoculation of mice with TK.sup.- and TK.sup.+ recombinant vaccinia virus (WR strain), significantly less dissemination of virus to other locations, including the brain, has been found (Taylor et al., 1991a).
Another enzyme involved with nucleotide metabolism is ribonucleotide reductase. Loss of virally encoded ribonucleotide reductase activity in herpes simplex virus (HSV) by deletion of the gene encoding the large subunit was shown to have no effect on viral growth and DNA synthesis in dividing cells in vitro, but severely compromised the ability of the virus to grow on serum starved cells (Goldstein et al., 1988). Using a mouse model for acute HSV infection of the eye and reactivatable latent infection in the trigeminal ganglia, reduced virulence was demonstrated for HSV deleted of the large subunit of ribonucleotide reductase, compared to the virulence exhibited by wild type HSV (Jacobson et al., 1989).
Both the small (Slabaugh et al., 1988) and large (Schmitt et al., 1988) subunits of ribonucleotide reductase have been identified in vaccinia virus. Insertional inactivation of the large subunit of ribonucleotide reductase in the WR strain of vaccinia virus leads to attenuation of the virus as measured by intracranial inoculation of mice (Child et al., 1990).
The vaccinia virus hemagglutinin gene (HA) has been mapped and sequenced (Shida, 1986). The HA gene of vaccinia virus is nonessential for growth in tissue culture (Ichihashi et al., 1971). Inactivation of the HA gene of vaccinia virus results in reduced neurovirulence in rabbits inoculated by the intracranial route and smaller lesions in rabbits at the site of intradermal inoculation (Shida et al., 1988). The HA locus was used for the insertion of foreign genes in the WR strain (Shida et al., 1987), derivatives of the Lister strain (Shida et al., 1988) and the Copenhagen strain (Guo et al., 1989) of vaccinia virus. Recombinant HA.sup.- vaccinia virus expressing foreign genes have been shown to be immunogenic (Guo et al., 1989; Itamura et al., 1990; Shida et al., 1988; Shida et al., 1987) and protective against challenge by the relevant pathogen (Guo et al., 1989; Shida et al., 1987).
Cowpox virus (Brighton red strain) produces red (hemorrhagic) pocks on the chorioallantoic membrane of chicken eggs. Spontaneous deletions within the cowpox genome generate mutants which produce white pocks (Pickup et al., 1984). The hemorrhagic function (u) maps to a 38 kDa protein encoded by an early gene (Pickup et al., 1986). This gene, which has homology to serine protease inhibitors, has been shown to inhibit the host inflammatory response to cowpox virus (Palumbo et al., 1989) and is an inhibitor of blood coagulation.
The u gene is present in WR strain of vaccinia virus (Kotwal et al., 1989b). Mice inoculated with a WR vaccinia virus recombinant in which the u region has been inactivated by insertion of a foreign gene produce higher antibody levels to the foreign gene product compared to mice inoculated with a similar recombinant vaccinia virus in which the u gene is intact (Zhou et al., 1990). The u region is present in a defective nonfunctional form in Copenhagen strain of vaccinia virus (open reading frames B13 and B14 by the terminology reported in Goebel et al., 1990a,b).
Cowpox virus is localized in infected cells in cytoplasmic A type inclusion bodies (ATI) (Kato et al., 1959). The function of ATI is thought to be the protection of cowpox virus virions during dissemination from animal to animal (Bergoin et al., 1971). The ATI region of the cowpox genome encodes a 160 kDa protein which forms the matrix of the ATI bodies (Funahashi et al., 1988; Patel et al., 1987). Vaccinia virus, though containing a homologous region in its genome, generally does not produce ATI. In WR strain of vaccinia, the ATI region of the genome is translated as a 94 kDa protein (Patel et al., 1988). In Copenhagen strain of vaccinia virus, most of the DNA sequences corresponding to the ATI region are deleted, with the remaining 3' end of the region fused with sequences upstream from the ATI region to form open reading frame (ORF) A26L (Goebel et al., 1990a,b).
A variety of spontaneous (Altenburger et al., 1989; Drillien et al., 1981; Lai et al., 1989; Moss et al., 1981; Paez et al., 1985; Panicali et al., 1981) and engineered (Perkus et al., 1991; Perkus et al., 1989; Perkus et al., 1986) deletions have been reported near the left end of the vaccinia virus genome. A WR strain of vaccinia virus with a 10 kb spontaneous deletion (Moss et al., 1981; Panicali et al., 1981) was shown to be attenuated by intracranial inoculation in mice (Buller et al., 1985). This deletion was later shown to include 17 potential ORFs (Kotwal et al., 1988b). Specific genes within the deleted region include the virokine N1L and a 35 kDa protein (C3L, by the terminology reported in Goebel et al., 1990a,b). Insertional inactivation of N1L reduces virulence by intracranial inoculation for both normal and nude mice (Kotwal et al., 1989a). The 35 kDa protein is secreted like N1L into the medium of vaccinia virus infected cells. The protein contains homology to the family of complement control proteins, particularly the complement 4B binding protein (C4bp) (Kotwal et al., 1988a). Like the cellular C4bp, the vaccinia 35 kDa protein binds the fourth component of complement and inhibits the classical complement cascade (Kotwal et al., 1990). Thus the vaccinia 35 kDa protein appears to be involved in aiding the virus in evading host defense mechanisms.
The left end of the vaccinia genome includes two genes which have been identified as host range genes, K1L (Gillard et al., 1986) and C7L (Perkus et al., 1990). Deletion of both of these genes reduces the ability of vaccinia virus to grow on a variety of human cell lines (Perkus et al., 1990).
Two additional vaccine vector systems involve the use of naturally host-restricted poxviruses, avipoxviruses. Both fowlpoxvirus (FPV) and canarypoxvirus (CPV) have been engineered to express foreign gene products. Fowlpox virus (FPV) is the prototypic virus of the Avipox genus of the Poxvirus family. The virus causes an economically important disease of poultry which has been well controlled since the 1920's by the use of live attenuated vaccines. Replication of the avipox viruses is limited to avian species (Matthews, 1982b) and there are no reports in the literature of avipoxvirus causing a productive infection in any non-avian species including man. This host restriction provides an inherent safety barrier to transmission of the virus to other species and makes use of avipoxvirus based vaccine vectors in veterinary and human applications an attractive proposition.
FPV has been used advantageously as a vector expressing antigens from poultry pathogens. The hemagglutinin protein of a virulent avian influenza virus was expressed in an FPV recombinant (Taylor et al., 1988a). After inoculation of the recombinant into chickens and turkeys, an immune response was induced which was protective against either a homologous or a heterologous virulent influenza virus challenge (Taylor et al., 1988a). FPV recombinants expressing the surface glycoproteins of Newcastle Disease Virus have also been developed (Taylor et al., 1990; Edbauer et al., 1990).
Despite the host-restriction for replication of FPV and CPV to avian systems, recombinants derived from these viruses were found to express extrinsic proteins in cells of nonavian origin. Further, such recombinant viruses were shown to elicit immunological responses directed towards the foreign gene product and where appropriate were shown to afford protection from challenge against the corresponding pathogen (Tartaglia et al., 1993 a,b; Taylor et al., 1992; 1991b; 1988b).
Thus, heretofore, the nucleotide and amino acid sequences for the CHV gB, gC and gD glycoproteins, have not been taught or suggested and, providing these sequences would be of great value. Further, vaccine, antigenic or immunological compositions from the nucleotides for the CHV gB, gC and gD glycoproteins (such as from vector systems containing such nucleotides) as well as from the glycoproteins themselves (such as from expression by the vector systems) have not heretofore been taught or suggested and, these nucleotides, vector systems, glycoproteins and compositions would be of great value.